Immunoassay test device with two fluid flow paths for detection and differentiation of two or more analytes

ABSTRACT

A device for determining presence or absence of infection due to an infectious agent is described. The device comprises a sample receiving zone configured to receive a liquid sample from a subject suspected of having an infection due to an infectious agent, the sample receiving zone positioned to distribute the sample along a first fluid flow path to a first label zone and along a second fluid flow path to a second label zone. Test lines in each fluid flow path capture a mobile detectable species as an indicator of presence or absence of the infectious agent. The device also comprises a reference line positioned in the one of the fluid flow paths. The bidirectional fluid flow paths emanate from a common sample zone and provide an efficient approach to detection and differentiate two species as indicators of the same infectious agent or as indicators of two different infectious agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/210,880, filed Aug. 27, 2015 and of U.S. Provisional Application No.62/268,455, filed Dec. 16, 2015, and of U.S. Provisional Application No.62/271,101, filed Dec. 22, 2015, each of which is incorporated herein byreference in its entirety.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

A Sequence Listing is being submitted electronically via EFS in the formof a text file, created Aug. 25, 2016, and named“0418960942SequenceListing.txt” (9,251 bytes), the contents of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The subject matter described herein relates to lateral flow immunoassaysthat have two separate fluid flow paths to detect and discriminate twospecies of interest in a fluid sample.

BACKGROUND

Rapid lateral flow immunoassays test devices have an extensive historyof use in both the clinical and the home settings. These devices areused to test for a variety of analytes, such as hormones, proteins,urine or plasma components and the like. These devices generallycomprise a lateral flow test strip, such as nitrocellulose or filterpaper, a sample application area, test results area and an analytespecific binding reagent that is bound to some kind of detectable label,such as a colored particle, fluorescent or luminescent tag, or an enzymedetection system. The simplicity of such devices is a factor inmaintaining their use in the marketplace, and additional tests fordetection and differentiation of multiple analytes in a single sampleare desired.

BRIEF SUMMARY

The following aspects and embodiments thereof described and illustratedbelow are meant to be exemplary and illustrative, not limiting in scope.

In one aspect, a device for determining presence or absence of infectiondue to an infectious agent is provided. The device comprises a samplereceiving zone configured to receive a liquid sample from a subjectsuspected of having an infection due to an infectious agent, the samplereceiving zone positioned to distribute the sample along a first fluidflow path to a first label zone and along a second fluid flow path to asecond label zone. Each of the first and second label zones comprise,respectively, a first mobilizable, detectable species and a secondmobilizable, detectable species, each mobilizable, detectable speciesable to bind the infectious agent or to antibody against the infectiousagent. The device also comprises a first test line in the first fluidflow path positioned downstream of the first label zone, the first testline comprising an immobilized species with binding affinity for thefirst mobilizable detectable species. The device also comprises a secondtest line in the second fluid flow path positioned downstream of thesecond label zone, the second test line comprising an immobilizedspecies with binding affinity for the second mobilizable detectablespecies. The device also comprises a reference line positioned in thefirst fluid flow path and comprising an immobilized species with bindingaffinity for a detectable moiety deposited on the device upstream of thereference line.

In one embodiment, the first fluid flow path and the second fluid flowpath are at an angle selected from a straight angle (180°), an obtuseangle and an acute angle.

In another embodiment, the reference line in the first fluid flow pathis downstream of the first test line.

In yet another embodiment, the device further comprises an additionalreference line in the second fluid flow path. In still anotherembodiment, the additional reference line is downstream of the secondtest line.

The device, in other embodiments, is designed such that the first testline comprises a first immobilized species that directly binds antibodyagainst the infectious agent present in the liquid sample and thatspecifically binds a conjugate comprised of the mobilizable, detectablespecies in the first label zone and an antibody against the infectiousagent in the sample.

In other embodiments, the second test line comprises a secondimmobilized species that directly binds antibody against the infectiousagent present in the liquid sample and that specifically binds aconjugate comprised of the mobilizable, detectable species in the secondlabel zone and an antibody against the infectious agent in the sample.

In some embodiments, the first and second immobilized species are thesame.

In one embodiment, the mobilizable, detectable species in the firstlabel zone is an anti-IgM antibody. In another embodiment, the anti-IgMantibody is a nonhuman anti-human IgM antibody.

In another embodiment, the immobilized species in the first test linecomprises a plurality of antigens for B. burgdorferi with bindingaffinity for the human IgM antibody against B. burgdorferi.

In yet another embodiment, the mobilizable, detectable species secondlabel zone is an anti-IgG antibody. In another embodiment, the anti-IgGantibody is a nonhuman anti-human IgG antibody.

In still another embodiment, the immobilized species in the first testline comprises a plurality of antigens for B. burgdorferi with bindingaffinity for the human IgG antibody against B. burgdorferi.

In one embodiment, the mobilizable, detectable species comprises a labeldetectable optically. Examples include but are not limited to afluorescent or chemiluminescent marker or a detectable particle, such asa europium bead.

In one embodiment, the sample receiving zone is positioned between thefirst label zone and the second label zone.

In another embodiment, the substrate comprises a single sample receivingzone that is common to the first label zone and the second label zone.

In one embodiment, the first and second test lines are within a singleoptical path of an optical detector for inspection by an instrument. Thesingle optical path is, in one embodiment, a path along one axis thatintersects the first and second test lines.

In another embodiment, the sample receiving zone dispenses sample to thefirst label zone and the second label zone in essentially equal amountsand at essentially equal rates.

In another aspect, a device for determining presence of infection due toan infectious agent is provided. The device comprises (i) a samplereceiving zone configured to receive a liquid sample from a subjectsuspected of having an infection due to an infectious agent, the samplereceiving zone positioned to distribute the sample along a first fluidflow path to a first label zone and along a second fluid flow path to asecond label zone, each of said first and second label zones comprising,respectively, a first mobilizable, detectable species and a secondmobilizable, detectable species, each mobilizable, detectable speciesable to bind separate, distinct antibodies against the infectious agent;(ii) a first test line in the first fluid flow path positioneddownstream of the first label zone, the first test line comprising animmobilized species with binding affinity for the first mobilizabledetectable species; (iii) a second test line in the second fluid flowpath positioned downstream of the second label zone, the second testline comprising an immobilized species with binding affinity for thesecond mobilizable detectable species; (iv) a reference line positionedin the first fluid flow path and comprising an immobilized species withbinding affinity for a detectable moiety deposited on the deviceupstream of the reference line.

In one embodiment, the first mobilizable, detectable species is anon-human, anti-human IgM antibody or a Lyme antigen. In anotherembodiment, the second mobilizable, detectable species is a non-human,anti-human IgG antibody or a Lyme antigen.

In another embodiment, the immobilized species on the first test linewith binding affinity for the first mobilizable detectable species is anon-human, anti-human IgM antibody or a Lyme antigen.

In still another embodiment, the immobilized species on the second testline with binding affinity for the second mobilizable detectable speciesis a non-human, anti-human IgG antibody or a Lyme antigen.

In yet another embodiment, the reference line is positioned downstreamof the first test line.

In other embodiments, the reference line comprises an immobilizedspecies with binding affinity for a non-human, anti-human IgG antibodyor a non-human, anti-human IgM antibody.

In one embodiment, the reference line comprises an immobilized specieswith binding affinity for a substance not present in the liquid sample.

In another embodiment, two or more separate, distinct antibodies againstthe infectious agent are present in the liquid sample and are capable ofbinding the immobilized species on each of the first test line and thesecond test line and capable of binding one of the mobilizable,detectable species on the first label line or on the second label line.

In another embodiment, the separate, distinct antibodies against theinfectious agent are present in the liquid sample and are capable ofbinding the mobilizable, detectable species on the first label line toform a first conjugate and on the second label line to form a secondconjugate, and the immobilized species on the first test line hasbinding affinity for the first conjugate and the immobilized species onthe second test line has binding affinity for the second conjugate.

In one embodiment, the immobilized species on the first test line is anon-human, anti-human IgM antibody. In another embodiment, theimmobilized species on the first test line is a non-human, anti-humanIgG antibody.

In another aspect, a method for staging infection by a pathogenicBorrelia species, such as Borrelia burgdorferi (B. burgdorferi), isprovided. The method comprises depositing a fluid sample from a personsuspected of or at risk of having been exposed to the pathogenicBorrelia species on a device as described herein; and inspecting (usingan instrument) the first test line and the second test line for thepresence or absence of the mobilizable detectable species.

In one embodiment, the fluid sample is blood, cerebrospinal fluid orurine.

In another embodiment, the fluid sample is blood and is deposited in anamount of less than 50 μL.

In other embodiment, inspecting is performed in about 10 minutes or lessafter depositing the fluid sample.

In still other embodiments, depositing and inspecting providessensitivity to detect an IgM antibody response to B. burgdorferiexposure in greater than 70% of exposed subjects within 2 weeks ofexposure. In another embodiment, depositing and inspecting providessensitivity to detect an IgG antibody response to B. burgdorferiexposure in greater than 70% of exposed subjects within 2 weeks ofexposure.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

Additional embodiments of the present methods and compositions, and thelike, will be apparent from the following description, drawings,examples, and claims. As can be appreciated from the foregoing andfollowing description, each and every feature described herein, and eachand every combination of two or more of such features, is includedwithin the scope of the present disclosure provided that the featuresincluded in such a combination are not mutually inconsistent. Inaddition, any feature or combination of features may be specificallyexcluded from any embodiment of the present invention. Additionalaspects and advantages of the present invention are set forth in thefollowing description and claims, particularly when considered inconjunction with the accompanying examples and drawings.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1B are top views of two embodiments of a test device havingfirst and second fluid flow paths;

FIG. 2 illustrates a top view of a test device with bilateral fluid flowpaths and position of optical windows for inspection of the test andoptional reference lines;

FIG. 3 is a perspective view of a test device with bidirectional fluidflow paths for discrimination of two analytes in a sample.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: MKKNDQIVAAIALRGVA

SEQ ID NO: 2 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: CMKKDDQIAAAMVLRGMAKDGQFALK

SEQ ID NO: 3 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: MKKNDQIGAAIALRGVA

SEQ ID NO: 4 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: MKKDDQIAAAIALRGMA

SEQ ID NO: 5 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: MKKDDQIAAAMVLRGMAKDGQFALKD

SEQ ID NO: 6 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: MKKDDQIAAAIALRGMAKDGKFAVKD

SEQ ID NO: 7 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: VQEGVQQEGAQQP

SEQ ID NO: 8 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: PVVAESPKKP

SEQ ID NO: 9 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: CPVVAESPKKP

SEQ ID NO: 10 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: LCPVVAESPKKP

SEQ ID NO: 11 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia:YGQNWTNPENMVTSGPFKLKERIPNEKIVFEKNNK

SEQ ID NO: 12 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: MTLFLFISCNNSGKDGNTSA

SEQ ID NO: 13 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: KMTLFLFISCNNSGKDGNTSA

SEQ ID NO: 14 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: TILVNLLISCGLTGA

SEQ ID NO: 15 is a peptide antigen with binding affinity to an epitopefrom a pathogenic strain of Borrelia: KDLKNKILKIKKEATGKGVLFEAFTGLKTG

SEQ ID NO: 16 is a fusion peptide antigen with binding affinity toepitopes from a pathogenic strain of Borrelia:VQEGVQQEGAQQPGGGMTLFLFISCNNSGKDGNTSAGGGMKKNDQIVAAIALRGVA

SEQ ID NO: 17 is a fusion peptide antigen with binding affinity toepitopes from a pathogenic strain of Borrelia:VQEGVQQEGAQQPGGGMKKNDQIVAAIALRGVA

SEQ ID NO: 18 is a fusion peptide antigen with binding affinity toepitopes from a pathogenic strain of Borrelia:VQEGVQQEGAQQPGGGMKKDDQIAAAMVLRGMAKDGQFALKD

SEQ ID NO: 19 is a synthetic linker peptide: GGGG

SEQ ID NO: 20 is the amino acid sequence of the outer surface protein Cof Borreliella burgdorferi (GenBank Acc. No. WP_012686633.1).

SEQ ID NO: 21 is the amino acid sequence of the decorin-binding proteinA of Borreliella burgdorferi (GenBank Acc. No. WP_010890380.1).

DETAILED DESCRIPTION I. Definitions

Various aspects now will be described more fully hereinafter. Suchaspects may, however, be embodied in many different forms and should notbe construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey its scope to those skilled in theart.

Where a range of values is provided, it is intended that eachintervening value between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the disclosure. For example, if a range of 1 μm to 8μm is stated, it is intended that 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μmare also explicitly disclosed, as well as the range of values greaterthan or equal to 1 μm and the range of values less than or equal to 8μM.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference toa “polymer” includes a single polymer as well as two or more of the sameor different polymers, reference to an “excipient” includes a singleexcipient as well as two or more of the same or different excipients,and the like.

“Sample” is any material to be tested for the presence or amount of ananalyte of interest. Preferably, a sample is a fluid sample, preferablya liquid sample. Examples of liquid samples that may be tested using atest device include bodily fluids including blood, serum, plasma,saliva, urine, ocular fluid, semen, sputum, nasal discharge and spinalfluid.

A “test strip” can include one or more bibulous or non-bibulousmaterials. If a test strip comprises more than one material, the one ormore materials are preferably in fluid communication. One material of atest strip may be overlaid on another material of the test strip, suchas for example, filter paper overlaid on nitrocellulose. Alternativelyor in addition, a test strip may include a region comprising one or morematerials followed by a region comprising one or more differentmaterials. In this case, the regions are in fluid communication and mayor may not partially overlap one another. Suitable materials for teststrips include, but are not limited to, materials derived fromcellulose, such as filter paper, chromatographic paper, nitrocellulose,and cellulose acetate, as well as materials made of glass fibers, nylon,dacron, PVC, polyacrylamide, cross-linked dextran, agarose,polyacrylate, ceramic materials, and the like. The material or materialsof the test strip may optionally be treated to modify their capillaryflow characteristics or the characteristics of the applied sample. Forexample, the sample application region of the test strip may be treatedwith buffers to correct the pH or specific gravity of an applied urinesample, to ensure optimal test conditions.

II. Device

In a first aspect, a device for determining presence of infection due toan infectious agent is provided. Various embodiments of the device willbe described with reference to certain drawing figures.

A first embodiment of a device is shown in FIG. 1A. A test strip 10comprises a sample receiving zone 12 configured to receive a liquidsample. Typically, the sample is from a subject suspected of having aninfection due to an infectious agent, and examples of types of patientsamples and of infectious agents are described below. Sample receivingzone 12 is positioned to distribute the sample along a first fluid flowpath, indicated by arrow 14 in FIG. 1A, to a first label zone 16 andalong a second fluid flow path, indicated by arrow 18 in FIG. 1A, to asecond label zone 20. Each of the first and second label zones comprise,respectively, a first mobilizable, detectable species and a secondmobilizable, detectable species, where each mobilizable, detectablespecies is able to bind to the infectious agent or to antibody againstthe infectious agent, as will be described.

The first fluid flow path and the second fluid flow path as depicted inFIG. 1A are at a straight angle of 180°. In other embodiments, the firstfluid flow path and the second fluid flow path can be at an obtuse anglefrom one another or at an acute angle from one another.

Test strip 10 also comprises a first test line 22 in the first fluidflow path and positioned downstream of the first label zone (16). Thefirst test line comprises an immobilized species with binding affinityfor the first mobilizable detectable species. Binding affinity intendsindirect binding or direct binding between two species, such as directbinding of an antigen to an antibody or indirect binding of a secondaryantibody to a conjugate formed of a primary antibody and an antigen,where the secondary antibody and primary antibody have binding affinity.For example, in one embodiment, an antibody in the patient sample isindicative of presence of infection by an infectious agent, and theantibody in the patient sample binds a mobilizable, detectable speciescomprised of a non-human antibody with binding affinity for the antibodyin the patient sample or an antigen of or from the infectious agentindicative of the suspected infection.

Test strip 10 also comprises a second test line 24 in the second fluidflow path (18) and positioned downstream of the second label zone (20).The second test line comprises an immobilized species with bindingaffinity for the second mobilizable, detectable species present in thesecond label zone. Binding affinity intends indirect binding or directbinding between two species, as described in the preceding paragraph.

Test strip 10 also comprises a reference line 26 positioned in the firstfluid flow path (14) and comprising an immobilized species with bindingaffinity for a detectable moiety deposited on or formed on the deviceupstream of the reference line. In one embodiment, reference line 26 isdownstream of first test line 22. Optionally, and as depicted in FIG. 1Bwhere like elements are denoted by like numerical identifiers as in FIG.1A, the test strip comprises a second or additional reference line 28 inthe second fluid flow path (18). In one embodiment, the additionalreference line is downstream of the second test line 24.

As mentioned above, in one embodiment, the first test line comprises animmobilized species that directly binds an antibody present in thepatient sample, the antibody being one raised by the patient's immunesystem against the infectious agent of interest and suspected of beingthe cause of infection in the patient. In another embodiment, the firsttest line comprises an immobilized species that binds a conjugate formedon the test device, the conjugate comprised of (i) the mobilizable,detectable species in the first label zone and (ii) an antibody presentin the patient sample, the antibody being one raised by the patient'simmune system against the infectious agent of interest and suspected ofbeing the cause of infection in the patient.

The second test line, in various embodiments, comprises an immobilizedspecies that directly binds antibody against the infectious agent, theantibody being one raised by the patient's immune system against theinfectious agent of interest and suspected of being the cause ofinfection in the patient. In another embodiment, the second test linecomprises an immobilized species that binds a conjugate formed on thetest device, the conjugate comprised of (i) the mobilizable, detectablespecies in the second label zone and (ii) an antibody present in thepatient sample, the antibody being one raised by the patient's immunesystem against the infectious agent of interest and suspected of beingthe cause of infection in the patient.

An illustrative test strip with continuing reference to FIGS. 1A-1B isnow described. In this exemplary test strip, it is desired to determinewhether a subject is at risk of Lyme disease or has Lyme disease, or,alternatively, it is desired to determine if infection with a Borreliaspecies, such as but not limited to Borrelia burgdorferi, Borreliaafzelii, Borrelia garinii, Borellia japonica, is at an early stage or alate stage of infection. To achieve these desires, a test strip thatcomprises a single, individual sample receiving zone is provided, and apatient's sample is deposited on or in the sample receiving zone. Thesample receiving zone is common to two fluid flow paths on the teststrip, where a first fluid flow path carries a portion of the depositedsample in a first direction and a second fluid flow path carries asecond portion of the deposited sample in a second direction. In oneembodiment, the two fluid flow paths and their directions of fluid floware at 180° from each other, so that the paths are along a common axis.Each of the two fluid flow paths comprises a label zone that comprises amobilizable, detectable species. The mobilizable, detectable species inthe exemplary test strip for staging or detecting infection by a speciesin the Borrelia genus is, in a first embodiment, a non-human, anti-humanantibody that has or is associated with a detectable label. Thenon-human, anti-human antibody is, in some embodiments, a non-human,anti-human IgG antibody bearing a detectable label, such as afluorescent, chemiluminescent, or other optically detectable tag, suchas a bead or chemical moiety. The non-human, anti-human antibody is, insome embodiments, a non-human, anti-human IgM antibody bearing adetectable label, such as a fluorescent, chemiluminescent, or otheroptically detectable tag, such as a bead or chemical moiety. In oneembodiment, optically detectable intends optically detectable by aninstrument and not visually detectable by an unaided human eye.

In this exemplary test strip, the detectable non-human, anti-human IgMantibody is deposited on the first label line, and the detectablenon-human, anti-human IgG antibody is deposited on the second labelline. More specifically, a detectable goat anti-human IgM antibody isdeposited on the first label line and a detectable goat anti-human IgGantibody is deposited on the second label line. Non-human, anti-humanIgG and IgM antibodies are exemplified as goat, anti-human antibodies,however the non-human portion of the antibody can be any mammal,including but not limited to mouse, rabbit, rat, sheep, etc.

Deposited on the first test line of the exemplary test strip fordetecting or staging Lyme disease is an antigen for a species in theBorrelia genus. For example, for detecting or staging Lyme infection dueto B. burgdorferi, one or more peptide antigens from B. burgdorferi isdeposited on the test lines in each of the flow paths on the test strip.In one example, peptide antigens with binding affinity to the OspC, C6,C10, FlaB or BBK07 regions of B. Burgdorferi are deposited in animmobile fashion to the test lines. In another embodiment, a recombinantprotein with binding affinity to B. Burgdorferi or to an antibodyagainst B. Burgdorferi is deposited on a test line. For example, therecombinant protein for the outer surface protein C (OspC) of B.Burgdorferi (GenBank Acc. No. WP_012686633.1; SEQ ID NO: 20), for thedecorin-binding protein A (DbpA) of B. Burgdorferi (GenBank Acc. No.WP_010890380.1; SEQ ID NO: 21), or fragments of these proteins that havebetween 12-60, 12-50, 12-40, 10-40 or 10-25 contiguous amino acidresidues from the protein are contemplated, as exemplified by SEQ ID NO:12 and SEQ ID NO: 14. Other examples of peptide antigens are known inart, such as in U.S. Pat. Nos. 8,338,556; 6,716,574; 6,719,983;8,071,109; 8,354,240; 6,475,492; 6,660,274; 7,887,815, 5,643,733, andU.S. Patent Publication No. 2015/0017666, which are each incorporated byreference herein.

In one embodiment, the peptide antigen deposited on one or both of thetest lines binds the C6 region of B. burgdorferi and has a sequenceselected from the exemplary sequences set forth as SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. Inother embodiments, the peptide antigen deposited on one or both of thetest lines comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 (or a peptide having 80%, 85%, 86%,87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity thereto) and is attached to the test line with abiotin-strepavidin interaction. In one embodiment, the peptide antigensor plurality of peptide antigens deposited on the first test line and onthe second test line are the same. In one embodiment, the peptideantigen deposited on one or both of the test lines has 80%, 85%, 86%,87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to one of the peptide or protein sequences disclosed herein.

The test strips described herein can be used in conjunction with ahousing member that encases the test strip. The housing member has oneor more apertures, the aperture(s) situated over the sample reservoirand the test lines and the reference line. Materials for housing membersare known in the art, as are materials for the test strip itself. FIG. 2illustrates a top view of a test device where optical windows in thehousing member are disposed over the test lines (22, 24) and referencelines (26, 28). Optical window 30 and optical window 32 define anopening visible to a user or an optical reader in an instrument forinterrogation of the test and reference lines.

FIG. 3 is a perspective view of a test device with bidirectional fluidflow paths for discrimination of two analytes in a sample. In thisembodiment, the test device has a base nitrocellulose layer 40 on whichthe test and control lines are deposited, such as test lines 42, 44 andcontrol lines 46, 48. A sample pad 50 is centrally positioned fordistribution of sample to each of the test and control lines via a labelpad, such as label pads 52, 54. The label pads are of a materialsuitable for receiving the mobilizable species and situated to overlapwith the nitrocellulose layer and the sample pad to provide contiguousfluid communication.

III. Methods of Use

The device described herein is contemplated for use in detection of anypathogenic or infectious agent, and several examples are now described.

A. Detection of Lyme Disease

Lyme disease is transmitted by the bite of various species of Ixodesticks carrying the etiologic agent, a pathogenic Borrelia bacterium (aspirochete). Organisms of the Borrelia burgdorferi sensu lato groupbelong to the family Spirochaetaceae, genus Borrelia. There are at least11 species in the B. burgdorferi complex and an unknown but large numberof substrains. At least three genospecies of the Borrelia burgdorferisensu lato group have been identified as pathogens: B. burgdorferi sensustricto, B. afzelli, and B. garinii. A current basis for diagnosis inthe absence of erythema migrans is the demonstration of an antibodyresponse against a pathogenic Borrelia. The test strip described hereinprovides a sensitive and specific diagnostic method for the detection ofLyme disease, e.g. at early times after infection, and provides a meansto stage the infection as early or late stage.

In one embodiment, a test strip with a plurality of peptides immobilizedon the first and second test lines in each of the fluid flow paths ormobilizable on the first and second label lines in each of the fluidflow paths is provided. The plurality of peptides can be the same ordifferent in each of the test lines and/or label lines. In oneembodiment, the plurality of peptides comprises 3 or more, or 4 or more,or 5 or more, or 6 or more, or 7 or more, or 8 or more, differentpeptide sequences from a Borellia species, such as B. burgdorferi sensulato. In another embodiment, the plurality of peptides comprises morethan 2 but 10 or fewer, or more than 2 but 9 or fewer, or more than 2but 8 or fewer, or more than 2 but 7 or fewer, or more than 2 but 6 orfewer, or more than 2 but 5 or fewer, or more than 2 but 4 or fewer,different peptides that bind specifically to an antibody against apathogenic Borellia species, such as B. burgdorferi sensu lato. In oneembodiment, the peptides are any combination of peptides with binding toOppA, Bbk32, OspC-typeK, RecA, BmpA, DbpA, ErpP, p35, OspF, CRASP 2,FlilB, p66, OspC-typeA, FlaB, or DdpB. In another embodiment, theplurality of peptide antigens includes a peptide that comprises anepitope from Borrelia flagellin p41 (e.g., a peptide having the sequenceVQEGVQQEGAQQP (SEQ ID NO: 7)), and/or an epitope from Borrelia OspC(e.g., a peptide having the sequence PVVAESPKKP (SEQ ID NO: 8)),including active (i.e., those that specifically bind) variants thereof.Alternatively, or in addition, the plurality of peptide antigensincludes a peptide that comprises an epitope from the VLsE (region IR6)Borrelia protein (e.g. the 26 amino acid peptide CMKKDDQIAAAMVLRGMAKDGQFALK (SEQ ID NO: 2)), or a shorter peptide from this region,such as a peptide with 12-18 contiguous resides from SEQ ID NO: 2 or12-18 non-contiguous residues from SEQ ID NO: 2, exemplified by thepeptide MKKNDQIGAAIALRGVA (SEQ ID NO: 3), or active variants thereof.

The antigen anchored to the test strip can also be a fusion peptide ofone, two, three, four or more peptides that bind to an antibody againsta pathogenic Borellia species, such as B. burgdorferi sensu lato. Askilled artisan can appreciate the variety of possible combination andthe variety of linkers to join the peptides. Examples are providedherein where, for example, a FlaB peptide (p41, SEQ ID NO: 7) isattached by a linker to a C6 peptide (SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6) to form a fusionpeptide as exemplified by SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:18. Another example is set forth herein as SEQ ID NO: 16 which consistsof a peptide from the p41 (FlaB) region (SEQ ID NO: 2) linked to apeptide from the OspC1 region (SEQ ID NO: 12) linked to a peptide forthe C6 region (SEQ ID NO: 1), the three peptides joined by a linkercomprised of three amino acid residues (GGG). In still otherembodiments, the linker peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more amino acids. In yet other embodiments, the linker comprises 1 to2, 1 to 5, 2 to 5, 2 to 4, 1 to 10, 5 to 10, 3 to 6, or 2 to 10 aminoacids. In still other embodiments, the linker is G, GG, GGG, or GGGG(SEQ ID NO: 19).

Suitable linkers for joining any of the peptide sequences disclosedherein can be readily selected and can be of any of a suitable ofdifferent lengths, such as from 1 amino acid (e.g., Gly) to 40 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids. In someembodiments, the linker comprises only glycines. In other embodiments,1, 2 3 or 4 of the glycines are substituted with serines.

Non-peptide linker moieties can also be used to join or link a carriermoiety to a mitochondrial fusion modulatory peptide. The linkermolecules are generally about 6-50 atoms long. The linker molecules mayalso be, for example, aryl acetylene, ethylene glycol oligomerscontaining 2-10 monomer units, diamines, diacids, amino acids, orcombinations thereof. Other linker molecules which can bind topolypeptides may be used in light of this disclosure.

It will be appreciated that the peptides disclosed herein and referencedby incorporation herein are merely exemplary of the peptide sequencesthat can be used in an assay. Embodiments of the assay contemplate useof peptide sequences that have 80%, 85%, 86%, 87%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of thesequences disclosed herein, when the two sequences are compared alongtheir entire length. It will also be appreciated that any of thesequences may be biotinylated, including biotinylated at the N-terminus,the C-terminus, or at an amine residue in an amino acid within the firstone, two, three, four, five amino acids (counting from the N-terminalamino acid) of the peptide. It will also be appreciated that any of thesequence may be modified at the N-terminus, the C-terminus, or both witha reactive moiety or a chemical linking moiety, such as a hydroxyl,carboxyl, amine or group.

Examples 1-4 relate to test strips for detection of IgG and IgMantibodies against a pathogenic Borrelia species (Examples 1-3) and forstaging as early state or late stage an infection by a pathogenicBorrelia species (Example 4). The test strips are designed to detect thepresence of immunoglobulins raised by a human subject infected with apathogenic Borrelia species. After infection, IgM antibodies developover a three to six week period, and this is referred to in the art, andherein, as an early stage infection. IgG antibodies develop within 4-12weeks after infection, and are indicative of infection at a later stage,and this is referred to herein as late stage infection. Accordingly, anearly stage of infection or early stage Lyme disease is indicated bypresence of IgM antibodies against a pathogenic Borrelia species in ablood sample taken 3-6 weeks after infection, and a late stage of Lymedisease is indicated by presence of IgG antibodies against a pathogenicBorrelia species in a blood sample taken 4-12 weeks after infection. Inone embodiment, a late state of Lyme disease is additionally indicatedby an amount of IgG antibodies in the sample that is greater than theamount of IgM antibodies in the sample. The test strip of Example 1comprises on the first and second label lines a conjugate of afluorescent label and a non-human (e.g., goat) antihuman immunoglobulinof IgM or IgG specificity against the pathogenic Borrelia species. Theblood immunoglobulins in the sample travel along the first and secondfluid flow paths to the first and second label lines. At each line, theblood immunoglobulin with specificity for the fluorescent-conjugate goatantihuman immunoglobulin of IgG or IgM binds to the conjugate to form amobile complex of a specific blood immunoglobulin (IgG or IgM) and afluorescent conjugate of the antihuman IgG or IgM. The complexes traveldownstream to their respective first or second test line, where theblood immunoglobulin portion of the complex binds with the peptideantigens deposited on each of the test lines, thus detecting on one testline blood IgG immunoglobulins for the pathogenic Borrelia species andon the other test line blood IgM immunoglobulins for the pathogenicBorrelia species.

The test strip of Example 1 is intended to interact with an instrumentwith an optical reader and software to operate the optical reader,gather signal emitted from the test strip and interpret the gatheredsignal data. The test strip includes a reference line used by theinstrument as a positional control for determination of the position ofthe first and second test lines. In the test strip embodiment of Example1, the reference line comprises an immobilized non-human antihumanimmunoglobulin specific for IgM (although an immobilized non-humanantihuman immunoglobulin specific for IgG could also be used). Theimmobilized non-human (e.g., rabbit) antihuman IgM antibody binds themobilizable non-human (e.g., goat) antihuman IgM antibody-detectableconjugate deposited on the label line and binds the complex formed onthe label line, the complex of the blood immunoglobulin IgM and thenon-human (e.g., goat) antihuman IgM antibody-detectable conjugate.Thus, in this embodiment, the reference line captures the complexcomprising the analyte of interest, the blood immunoglobulin raisedagainst the pathogen of interest (in this case, a species of Borrelia).For this reason, the reference line is positioned downstream of the testline since if a small amount of the analyte of interest to be detectedis present, it would be captured on the reference line and the teststrip would return a false negative. A reference line capable ofcapturing two detectable moieties on the test strip—in this case boththe complex formed on the strip and the detectable label species on thelabel line—increases the detectable signal on the reference line.

An alternative embodiment of a test strip for determining the presenceof an immunoglobulin against a pathogenic Borrelia species, and thusindicative of the presence or absence of Lyme disease, is described inExample 2. In this test strip, the label line comprises mobilizable,detectable conjugate of one or more peptide antigens with bindingspecificity to immunoglobulins IgG or IgM in the blood raised againstthe pathogenic Borrelia species and a detectable label. The detectableconjugate binds the blood immunoglobulin, forming a complex that travelsdownstream to the test line. One test line on the test strip comprises aspecies with binding affinity to IgG immunoglobulin and the other testline comprises a species with binding affinity to IgM immunoglobulin. Inthis way, IgG and IgM are discriminated on the test strip at the testlines. In the embodiment of Example 1, IgG and IgM are discriminated onthe test strip at the label lines. In the test strip of Example 2, abinding pair that is independent of the antigen-antibody pair associatedwith the pathogenic Borrelia species is used on the reference line.Examples include horse radish peroxidase and anti-horse radishperoxidase and glucose oxidase and anti-glucose oxidase, where one ofthe binding pair is immobilized on the reference line and the otherbinding member of the pair is deposited on the test strip upstream ofthe reference line. Accordingly, test devices with a reference line thatcomprises an immobilized species with binding affinity for a substancenot present in the liquid sample are contemplated.

Another embodiment of a test strip for determining the presence of animmunoglobulin against a pathogenic Borrelia species, and thusindicative of the presence or absence of Lyme disease, is described inExample 3. One test line on the test strip comprises a species withbinding affinity to IgG immunoglobulin and the other test line comprisesa species with binding affinity to IgM immunoglobulin. In this way, IgGand IgM are discriminated on the test strip. On one side of the samplereservoir (e.g., see FIG. 1A) in a downstream to upstream direction aredeposited a first label line comprising mobilizable goat anti-human IgMantibodies attached to europium beads. Downstream from this first labelline is a first test line with immobilized peptide antigens for the OspCregion (e.g., peptide antigens identified as SEQ ID NO: 12 or SEQ ID NO:13) and the DbpA region (e.g., peptide antigen identified as SEQ ID NO:14) of B. burgdorferi. A reference line with immobilized rabbitanti-goat IgM antibodies is downstream of the first label line. On theopposing side of the sample reservoir (e.g., see FIG. 1A) in adownstream to upstream direction are deposited a second label linecomprising mobilizable mouse anti-human IgG antibodies bound to europiumbeads and a second test line with immobilized peptide antigens for theC10 (SEQ ID NO: 8 or SEQ ID NO: 10), C6 (SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6) and p41 (FlaB; SEQID NO: 7) epitopes of B. burgdorferi. The peptide antigens were modifiedto include a biotin moiety at the N-terminus or in the N-terminal regionand anchored to the test strip via binding with an anchored streptavidinmolecule.

It can also be appreciated from the exemplary test strips that hereinprovide a means to differentiate separate, distinct antibodies raisedagainst the same infectious agent. The test strips comprise animmobilized species on each of the first test line and the second testline that bind each of the separate, distinct antibodies raised againstthe same infectious agent, and discriminate the two separate, distinctantibodies by the use of a mobilizable, detectable species specific forone of the separate, distinct antibodies on the first label line and onthe second label line.

As mentioned above, the tests strip of Example 1-3 can be used to stageas early stage or late stage an infection by a pathogenic Borreliaspecies. As described in Example 4, the method comprises depositing afluid sample from a person suspected of or at risk of having beenexposed to the pathogenic Borrelia species on a device as describedherein, and inspecting the first test line and the second test line forthe presence or absence of the mobilizable detectable species. In oneembodiment, depositing and inspecting provides sensitivity to detect anIgM antibody response to B. burgdorferi exposure in greater than 70% ofexposed subjects within 2 weeks of exposure. In another embodiment,depositing and inspecting provides sensitivity to detect an IgG antibodyresponse to B. burgdorferi exposure in greater than 70% of exposedsubjects within 2 weeks of exposure.

In another study, detailed in Example 5, a test strip as depicted inFIG. 1A was prepared by positioning a common, single sample padcentrally on a strip of nitrocellulose. To one side of the single,common sample pad in a downstream to upstream direction was deposited aplurality of detectable markers (e.g., europium beads) with surfacebound goat anti-human IgM antibodies to form a first label line.Downstream of the first label line a first test line was deposited, thefirst test line composed of recombinant OspC (SEQ ID NO: 20) andrecombinant DbpA (SEQ ID NO: 21), each linked at its N-terminus tobiotin and immobilized to the nitrocellulose via streptavidin.Downstream of the first test line was deposited a rabbit anti-goat IgGantibody to form a reference line on the nitrocellulose. On the opposingside of the common, single sample pad, in a downstream to upstreamdirection, was deposited a plurality of detectable markers (e.g.,europium beads) with surface bound mouse anti-human IgG antibodies toform a second label line. Downstream of the second label line a secondtest line was deposited, the second test line composed immobilizedpeptide antigens for the C10 (SEQ ID NO: 8), C6 (SEQ ID NO: 6) andC6-p41 (SEQ ID NO: 18) epitopes of B. burgdorferi. The peptide antigenswere modified to include a biotin moiety at the N-terminus or in theN-terminal region and anchored to the nitrocellulose substrate viabinding with an anchored streptavidin molecule. The C10 peptide antigenwas modified at its C-terminus with a hydroxyl group. An absorbent padwas positioned on each end of the test strip.

Blood samples from individuals presenting with acute erythema migrans(an early stage indication of Lyme disease) or with Lyme arthritis (anindicator of late stage Lyme disease) were obtained, as well as bloodsamples from healthy individuals to serve as a negative control. Thesamples were diluted with buffer and a known volume was placed on thesample pad of the test strip. Each test strip was incubated and theninserted into an instrument with an optical system and software tointerrogate the test lines and the reference lines for presence orabsence of the detectable markers.

The results from the instrument-read test strips were interpreted andcompared to results from enzyme immunoassay (EIA), Western Blot, and thetwo-tiered testing for Lyme disease recommended by the Center forDisease Control and Prevention. Of the eight (8) early Lyme erythemamigrans samples, both the instrument-read test strips for IgM detectionand the Western Blot IgM detected ⅝ (62.5%), and the samples tested byEIA for IgM detected 4/8 (50%), suggesting that the instrument-read teststrip is more sensitive than EIA for early stage Lyme detection.Accordingly, in one embodiment, a test strip as described hereinprovides a sensitivity for detection of IgM that is greater than thatprovided by enzyme immunoassay, for detection of Lyme disease or forstaging Lyme disease as early stage or late stage.

Of the four (4) late Lyme samples tested, the instrument-read teststrips for IgG, Western Blot IgG and EIA detected all of the samples(100%) suggesting that the instrument-read test strip for IgG hassensitivity equivalent to detection of IgG via Western Blot and EIA.Accordingly, in one embodiment, a test strip as described hereinprovides a sensitivity for detection of IgG that is essentially the sameas that provided by Western blot and/or by enzyme immunoassay, fordetection of Lyme disease or for staging Lyme disease as early stage orlate stage.

With regard to the control samples, the instrument-read test strip ofIgM and IgG and Western Blot IgM and IgG were negative for all twenty(20) negative control samples tested (100% negative agreement). The EIAreported four (4) samples positive (80% negative agreement). Thisdemonstrates that the instrument-read test strip for IgM and IgG is morespecific than EIA and equivalent to IgM and IgG Western Blot.Accordingly, in one embodiment, a test strip as described hereinprovides a specificity for detection of IgM and/or IgG that is greateror higher than that provided by enzyme immunoassay, for detection ofLyme disease or for staging Lyme disease as early stage or late stage.

B. Other Exemplary Test Strips for Detection and Discrimination of Twoor More Species

In addition to the test strip described above for detection anddifferentiation of IgG and IgM immunoglobulins against a pathogenicBorrelia species, test devices that detect and differentiate ordiscriminate herpes simplex virus-1 and herpes simplex virus-2 (HSV-1and HSV-2), influenza A and influenza B (Flu A and Flu B), influenza A+Band respiratory syncytial virus (RSV) are contemplated. Alsocontemplated is a test strip for detection and differentiation of IgAand IgG associate with Zika virus. As can be appreciated, thebidirectional test strip with two separate fluid flow pathscommunicating from a common sample reservoir provide an approach todifferentiating the two or more analytes of interest from a sampleplaced on the common sample reservoir.

With regard to a test device for detection and differentiation of HSV-1and HSV-2, a test strip is contemplated that is comprised of a firstlabel line and a second label line each comprising a mobilizable,detectable anti-human IgG antibody. The first test line on the teststrip comprises an immobilized antigen with binding affinity for HSV-1and the second test line on the test strip comprises an immobilizedantigen with binding affinity for HSV-2. The reference line isdownstream of the first test line and comprises a binding member of abinding pair independent from the HSV infectious pathogen or comprises anon-human antibody that binds the mobilizable, detectable anti-human IgGantibody deposited on the label lines.

With regard to a test device for detection and differentiation of Flu Aand Flu B, a test strip is contemplated that is comprised of a firstlabel line with a mobilizable, detectable anti-flu A nucleoproteinantibody and a second label line with a mobilizable, detectable anti-fluB nucleoprotein antibody. The first test line and the second test lines,respectively, comprise an immobilized anti-flu A nucleoprotein antibodyand an immobilized anti-flu B nucleoprotein antibody. The reference lineis downstream of the first test line and comprises a binding member of abinding pair independent from the Flu A, Flu B infectious pathogen orcomprises a non-human antibody that binds the mobilizable, detectableanti-flu A (or flu B) nucleoprotein antibody deposited on the labelline.

With regard to a test device for detection and differentiation of FluA/B and RSV, a test strip is contemplated that is comprised of a firstlabel line with a mobilizable, detectable anti-flu A/B nucleoproteinantibody and a second label line with a mobilizable, detectable anti-RSVantibody. The first test line and the second test lines, respectively,comprise an immobilized anti-flu A/B nucleoprotein antibody and animmobilized anti-RSV antibody. The reference line is downstream of thefirst test line and comprises a binding member of a binding pairindependent from the infectious pathogens or interest or comprises anon-human antibody that binds the mobilizable, detectable antibodydeposited on a label line.

Accordingly, the device herein is designed to determine presence ofinfection due to an infectious agent, and is able to detect anddiscriminate two analytes (or two or more analytes) in a biologicalsample that are indicative of the infectious agent. The device comprisesa sample receiving zone configured to receive a liquid sample from asubject suspected of having an infection due to an infectious agent, thesample receiving zone positioned to distribute the sample along a firstfluid flow path to a first label zone and along a second fluid flow pathto a second label zone. Each of the first and second label zonescomprise, respectively, a first mobilizable, detectable species and asecond mobilizable, detectable species, each mobilizable, detectablespecies able to bind separate, distinct antibodies against theinfectious agent. The device also comprises a first test line in thefirst fluid flow path positioned downstream of the first label zone, thefirst test line comprising an immobilized species with binding affinityfor the first mobilizable detectable species. The device also comprisesa second test line in the second fluid flow path positioned downstreamof the second label zone, the second test line comprising an immobilizedspecies with binding affinity for the second mobilizable detectablespecies. The device also comprises a reference line positioned in thefirst fluid flow path and comprising an immobilized species with bindingaffinity for a detectable moiety deposited on the device upstream of thereference line.

In one embodiment, the volume of fluid sample deposited on the device isless than about 100 μL, preferably less than 75 μL, preferably less than50 μL, preferably between 10-75 μL, preferably between 10-60 μL andpreferably between 10-50 μL.

In another embodiment, the test yields a detectable signal at the firstand/or second test line within about 20 minutes or less after depositingthe fluid sample, or within about 15 minutes or less after depositingthe fluid sample, or within about 10 minutes or less after depositingthe fluid sample, or between about 10-30 minutes after depositing thefluid sample, or between about 10-45 minutes after depositing the fluidsample.

IV. Examples

The following examples are illustrative in nature and are in no wayintended to be limiting.

Example 1 Test Strip for Detecting Presence of Antibodies Against B.burgdorferi

A lateral flow immunoassay device for detection of the presence orabsence of IgG and IgM antibodies in the blood of a human is prepared asfollows. A test strip is constructed to have a centrally-positionedsample pad that serves as a common sample reservoir for the test strip.On one side of the sample reservoir (e.g., see FIG. 1A) in a downstreamto upstream direction are deposited a first label line comprising goatanti-human IgM antibodies attached to a europium bead, a first test linewith immobilized peptide antigens comprised of the peptide sequenceslisted below, and a reference line with immobilized rabbit anti-goat IgMantibodies. On the opposing side of the sample reservoir (e.g., see FIG.1A) in a downstream to upstream direction are deposited a second labelline comprising goat anti-human IgG antibodies bound to a europium beadand a second test line with immobilized peptide antigens comprised ofthe peptide sequences listed below. The peptide antigens immobilizedthrough a streptavidin moiety attached to the test strip and a biotinattached to each peptide and deposited in each of the first and secondtest lines are:

(SEQ ID NO: 4) MKKDDQIAAAIALRGMA (SEQ ID NO: 11)YGQNWTNPENMVTSGPFKLKERIPNEKIVFEKNNK (SEQ ID NO: 12)MTLFLFISCNNSGKDGNTSA;  and (SEQ ID NO: 16)VQEGVQQEGAQQPGGGMTLFLFISCNNSGKDGNTSAGGGMKKNDQIVAAI ALRGVA

An absorbent pad is positioned on each end of the test strip.

A blood sample from a human suspected of having Lyme disease caused byB. burgdorferi is deposited on the sample reservoir. 15 minutes later,the test strip is inserted into an instrument with an optical system andsoftware to determine presence or absence of europium in the first testline and in the second test line. The instrument uses signal from thereference line to determine position of the first and second test lines.

Example 2 Test Strip for Detecting Presence of Antibodies Against B.burgdorferi

A lateral flow immunoassay device for detection of the presence orabsence of IgG and IgM antibodies in the blood of a human is prepared asfollows. A test strip is constructed to have a centrally-positionedsample pad that serves as a common sample reservoir for the test strip.On one side of the sample reservoir (e.g., see FIG. 1A) in a downstreamto upstream direction are deposited a first label line comprising aplurality of peptide antigens from B. burgdorferi, each having specificbinding to antibodies against B. burgdorferi and attached to a europiumbead; a first test line with immobilized goat-anti-human IgG antibodies;and a reference line with immobilized anti-glucose oxidase antibodies.The first label line comprises glucose oxidase enzyme attached to aeuropium bead that travels downstream with the fluid sample. On theopposing side of the sample reservoir (e.g., see FIG. 1A) in adownstream to upstream direction are deposited a second label linecomprising a plurality of peptide antigens from B. burgdorferi, eachhaving specific binding to antibodies against B. burgdorferi andattached to a europium bead; and a second test line with immobilizedgoat-anti-human IgM antibodies. The peptide antigens deposited on thefirst and second label lines are:

(SEQ ID NO: 4) MKKDDQIAAAIALRGMA (SEQ ID NO: 11)YGQNWTNPENMVTSGPFKLKERIPNEKIVFEKNNK (SEQ ID NO: 12)MTLFLFISCNNSGKDGNTSA;  and (SEQ ID NO: 16)VQEGVQQEGAQQPGGGMTLFLFISCNNSGKDGNTSAGGGMKKNDQIVAAI ALRGVA

An absorbent pad is positioned on each end of the test strip.

A blood sample from a human suspected of having Lyme disease caused byB. burgdorferi is deposited on the sample reservoir. 15 minutes later,the test strip is inserted into an instrument with an optical system andsoftware to determine presence or absence of europium in the first testline and in the second test line. The instrument uses signal from thereference line to determine position of the first and second test lines.

Example 3 Test Strip for Detecting Presence of Antibodies Against B.burgdorferi

A lateral flow immunoassay device for detection of the presence orabsence of IgG and IgM antibodies in the blood of a human is prepared asfollows. A test strip is constructed to have a centrally-positionedsample pad that serves as a common sample reservoir for the test strip.On one side of the sample reservoir (e.g., see FIG. 1A) in a downstreamto upstream direction are deposited a first label line comprisingmobilizable goat anti-human IgM antibodies attached to europium beads, afirst test line with immobilized peptide antigens for the OspC region(SEQ ID NO: 12) and the DbpA region (SEQ ID NO: 14) of B. burgdorferi,and a reference line with immobilized rabbit anti-goat IgM antibodies.On the opposing side of the sample reservoir (e.g., see FIG. 1A) in adownstream to upstream direction are deposited a second label linecomprising mobilizable mouse anti-human IgG antibodies bound to europiumbeads and a second test line with immobilized peptide antigens for theC10 (SEQ ID NO: 8 or SEQ ID NO: 10), C6 (SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6) and p41 (FlaB; SEQID NO: 7) epitopes of B. burgdorferi. The peptide antigens were modifiedto include a biotin moiety at the N-terminus or in the N-terminal regionand anchored to the test strip via binding with an anchored streptavidinmolecule.

An absorbent pad is positioned on each end of the test strip.

A blood sample from a human suspected of having Lyme disease caused byB. burgdorferi is deposited on the sample reservoir. 15 minutes later,the test strip is inserted into an instrument with an optical system andsoftware to determine presence or absence of europium in the first testline and in the second test line. The instrument uses signal from thereference line to determine position of the first and second test lines.

Example 4 Test Strip for Staging Infection by B. burgdorferi

A test strip as described in Example 1 is prepared. A blood sample istaken from a female subject presenting with symptoms of myalgia, fatigueand erythema migrans. The blood sample is deposited on the sample pad ofthe test strip. 20 minutes later, the test strip is inserted into aninstrument with an optical system and software to determine quantity orrelative quantities of IgG and IgM antibodies against B. Burgdorferi inthe blood sample. The test line where IgM antibodies against B.Burgdorferi in the blood sample are captured emits a detectable signalwhen probed by the instrument. The test line where IgG antibodiesagainst B. Burgdorferi in the blood sample are captured emits adetectable signal when probed by the instrument. The signal emitted atthe test line of IgG antibodies is greater than the signal emitted atthe test line of IgM antibodies, indicative of a later stage infectionas IgG antibodies are raised 4-12 weeks after infection (E. D. Shapiroand P. Auwaerter, INFECTIOUS DISEASE AND ANTIMICROBIAL AGENTS: Borreliaburgdorferi (Lyme Disease), 2002 Edition).

Example 5 Test Strip for Detecting Antibodies Against B. burgdorferi

A test strip was prepared as follows. A sample pad was centrallypositioned on a strip of nitrocellulose. To one side of the single,common sample pad (e.g., see FIG. 1A) in a downstream to upstreamdirection was deposited a plurality of europium beads with surface boundgoat anti-human IgM antibodies to form a first label line. Downstream ofthe first label line a first test line was deposited, the first testline composed of recombinant OspC (SEQ ID NO: 20) and recombinant DbpA(SEQ ID NO: 21), each linked at its N-terminus to biotin and immobilizedto the nitrocellulose via streptavidin. Downstream of the first testline was deposited a rabbit anti-goat IgG antibody to form a referenceline on the nitrocellulose. On the opposing side of the sample pad, in adownstream to upstream direction, was deposited a plurality of europiumbeads with surface bound mouse anti-human IgG antibodies to form asecond label line. Downstream of the second label line a second testline was deposited, the second test line composed immobilized peptideantigens for the C10 (SEQ ID NO: 8), C6 (SEQ ID NO: 6) and C6-p41 (SEQID NO: 18) epitopes of B. burgdorferi. The peptide antigens weremodified to include a biotin moiety at the N-terminus or in theN-terminal region and anchored to the nitrocellulose substrate viabinding with an anchored streptavidin molecule. The C10 peptide antigenwas modified at its C-terminus with a hydroxyl group. An absorbent padwas positioned on each end of the test strip.

Blood samples from individuals presenting with acute erythema migrans(an early stage indication of Lyme disease) or with Lyme arthritis (anindicator of late stage Lyme disease) were obtained, as well as bloodsamples from healthy individuals to serve as a negative control. Using acalibrated micropipette, blood samples were diluted 1:10 in a buffer(i.e., blood sample volume of 25 μL in 250 μL buffer). Using acalibrated micropipette, 100 μl of the diluted blood sample was added tothe sample port of a test device for contact with the sample pad. Eachtest strip was incubated for 10 minutes and then inserted into aninstrument with an optical system and software to interrogate the testlines and the reference lines for presence or absence of the detectableeuropium bead. The results from each test device run were interpreted aseither positive (above the assay cutoff) or negative (below the assaycutoff).

The results from the instrument read test strips were interpreted andcompared to results from enzyme immunoassay (EIA), Western Blot, and thetwo-tiered testing for Lyme disease recommended by the Center forDisease Control and Prevention. Of the eight (8) early Lyme erythemamigrans samples, both the instrument-read test strips for IgM detectionand the Western Blot IgM detected ⅝ (62.5%), and the samples tested byEIA for IgM detected 4/8 (50%), suggesting that the instrument-read teststrip is more sensitive than EIA for early stage Lyme detection.Accordingly, in one embodiment, a test strip as described hereinprovides a sensitivity for detection of IgM that is greater than thatprovided by enzyme immunoassay, for detection of Lyme disease or forstaging Lyme disease as early stage or late stage.

Of the four (4) late Lyme samples tested, the instrument-read teststrips for IgG, Western Blot IgG and EIA detected all of the samples(100%) suggesting that the instrument-read test strip for IgG hassensitivity equivalent to detection of IgG via Western Blot and EIA.Accordingly, in one embodiment, a test strip as described hereinprovides a sensitivity for detection of IgG that is essentially the sameas that provided by Western blot and/or by enzyme immunoassay, fordetection of Lyme disease or for staging Lyme disease as early stage orlate stage.

With regard to the control samples, the instrument-read test strip ofIgM and IgG and Western Blot IgM and IgG were negative for all twenty(20) negative control samples tested (100% negative agreement). The EIAreported four (4) samples positive (80% negative agreement). Thisdemonstrates that the instrument-read test strip for IgM and IgG is morespecific than EIA and equivalent to IgM and IgG Western Blot.Accordingly, in one embodiment, a test strip as described hereinprovides a specificity for detection of IgM and/or IgG that is greateror higher than that provided by enzyme immunoassay, for detection ofLyme disease or for staging Lyme disease as early stage or late stage.

There was 100% agreement between the instrument-read test strip for IgMand Western Blot IgM results across the positive and negative samples.There was 90.6% agreement between the instrument-read test strip for IgGand Western Blot IgG results. There was a 96.8% agreement between theinstrument-read test strip (IgM+IgG) results with the 2-Tier method.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

It is claimed:
 1. A device, comprising: only one sample receiving zone,the sample receiving zone configured to receive a liquid sample from asubject suspected of comprising an analyte of interest, the samplereceiving zone positioned to distribute the sample along a first fluidflow path to a first label zone and along a second fluid flow path,distinct from the first fluid flow path, to a second label zone, each ofsaid first and second label zones comprising, respectively, a firstmobilizable, detectable species that binds IgM antibodies specific tothe analyte of interest present in the liquid sample and a secondmobilizable, detectable species that binds IgG antibodies specific tothe analyte of interest present in the liquid sample; and only onereference line, wherein the reference line is positioned in the firstfluid flow path and comprises an immobilized species with bindingaffinity for a detectable moiety deposited on the device upstream of thereference line; wherein the device comprises a first test line in thefirst fluid flow path positioned downstream of the first label zone, thefirst test line comprising a first immobilized species that is capableof directly binding the IgM antibodies and that is capable ofspecifically binding a conjugate comprised of the first mobilizable,detectable species and the IgM antibodies, and wherein the firstimmobilized species comprises an antigen from the analyte of interest;and wherein the device comprises a second test line in the second fluidflow path positioned downstream of the second label zone, the secondtest line comprising a second immobilized species that is capable ofdirectly binding the IgG antibodies and that is capable of specificallybinding a conjugate comprised of the second mobilizable, detectablespecies and the IgG antibodies, and wherein the second immobilizedspecies comprises an antigen from the analyte of interest.
 2. The deviceof claim 1, wherein the first fluid flow path and the second fluid flowpath are at an angle selected from a straight angle (180°), an obtuseangle and an acute angle.
 3. The device of claim 1, wherein thereference line is downstream of the first test line.
 4. The device ofclaim 1, wherein the mobilizable, detectable species in the first labelzone comprises a nonhuman anti-human IgM antibody.
 5. The device ofclaim 4, wherein the immobilized species in the first test linecomprises a plurality of antigens for B. burgdorferi with bindingaffinity for the human IgM antibody against B. burgdorferi.
 6. Thedevice of claim 1, wherein the mobilizable, detectable species secondlabel zone comprises a nonhuman anti-human IgG antibody.
 7. The deviceof claim 6, wherein the immobilized species in the second test linecomprises a plurality of antigens for B. burgdorferi with bindingaffinity for the human IgG antibody against B. burgdorferi.
 8. Thedevice of claim 1, wherein the mobilizable, detectable species comprisesa label detectable optically.
 9. The device of claim 8, wherein theoptically detectable label is a fluorescent or chemiluminescent marker.10. The device of claim 1, wherein the sample receiving zone ispositioned between the first label zone and the second label zone. 11.The device of claim 1, further comprising a substrate, wherein thesample receiving zone, the first and second fluid flow paths, the firstand second label zones, the first and second test lines, and thereference line are deposited on the substrate for direct contacttherewith.
 12. The device of claim 1, wherein the first and second testlines are within a single optical window for inspection by aninstrument.
 13. The device of claim 1, wherein the sample receiving zonedispenses sample to the first label zone and the second label zone inessentially equal amounts or at essentially equal rates.
 14. A device,comprising: only one sample receiving zone, the sample receiving zoneconfigured to receive a liquid sample from a subject suspected ofcomprising an analyte of interest, the sample receiving zone positionedto distribute the sample along a first fluid flow path to a first labelzone and along a second fluid flow path, distinct from the first fluidflow path, to a second label zone, the first label zone comprises amobilizable, detectable nonhuman anti-human IgM antibody that binds IgMantibodies specific to the analyte of interest present in the liquidsample, the second label zone comprises a mobilizable, detectablenonhuman anti-human IgG antibody that binds IgG antibodies specific tothe analyte of interest present in the liquid sample; and only onereference line, wherein the reference line is positioned in the firstfluid flow path and comprises an immobilized species with bindingaffinity for a detectable moiety deposited on the device upstream of thereference line; wherein the device comprises a first test line in thefirst fluid flow path positioned downstream of the first label zone, thefirst test line comprising a first immobilized species that is capableof specifically binding a conjugate comprised of the first mobilizable,detectable species and the IgM antibodies specific to the analyte ofinterest, and wherein the first immobilized species comprises an antigenfrom the analyte of interest; and wherein the device comprises a secondtest line in the second fluid flow path positioned downstream of thesecond label zone, the second test line comprising a second immobilizedspecies that is capable of specifically binding a conjugate comprised ofthe second mobilizable, detectable species and the IgG antibodiesspecific to the analyte of interest, and wherein the second immobilizedspecies comprises an antigen from the analyte of interest.